All publications mentioned throughout this application are fully incorporated herein by reference, including all references cited therein.
Biomaterials are currently in use or under investigation as implants to facilitate restoration and regeneration of defective or missing tissues in conditions caused by disease, trauma or reconstructive surgical procedures. In particular, injectable biomaterials are ideal for tissue restoration since the flowable material may be delivered via a small incision, allowing minimally invasive access to the tissue space where appropriate. Fluids can interdigitate with the irregular cavity defects (e.g., following surgical procedure) and may, depending on the material used, physically bond to the adjacent tissue. Injectable biomaterials also allow for incorporation and uniform dispersion of cells and/or therapeutic agents, such as growth factors and cytokines that are valuable in enhancing the tissue repair processes.
Additional advantageous features of biomaterials for tissue reconstruction and restoration include their ease of production and handling compared to the processes involving cells and their being off-the-shelf products. Biomaterials, especially those derived from plant/algae, such as the alginate polysaccharides, are non immunogenic, biocompatible, biodegradable and not affected by age or disease. They are relatively low cost as compared to existing cell therapy approaches.
The primary considerations for injectable scaffolds for utilities such as bulking, filling voids and tissue reconstruction include mechanical strength and durability, promotion of tissue formation, biodegradability, biocompatibility, sterilizability, minimal setting time and temperature change, low viscosity for easy injection, as well as ease in accessing the defect. The scaffold must exhibit the necessary mechanical properties as well as provide physical support. Preferably, the scaffold would promote matrix formation while degrading over time. The biocompatibility of the material is also of great importance. Neither the initial material nor its degradation products should elicit an unresolved immune response, promote immunotoxicity, or express cytotoxicity. To minimize infections and related immune responses, the implanted material must be easily sterilized while retaining the original bioactivity and chemical composition.
The candidate biomaterials can be injected as viscous fluids and then cured by methods such as thermosensitive or pH-sensitive crosslinking, photopolymerization, or addition of a solidifying agent to form a gel-like substance. The biomaterials can be also implanted as pre-formed solid matrix, as hydrogel or macroporous scaffold.
The present invention describes a method for replacing or supplementing lost organ function using therapeutical biomaterials. Such method is advantageous over pharmacological manipulation or transplantation of whole organ or parts of organ, as the later do not cure a disease but rather modify its outcome, or trade the original disease for the complications of non-specific immunosuppression.
Every structure in living organisms is in a dynamic state of equilibrium, undergoing constant renewal, remodeling and replacement of functional tissue which vary from organ to organ and structure to structure. Following extensive damage, these abilities of remodeling and replacement are greatly impaired. Biomaterials provide temporary scaffoldings for remaining organ tissue cells, and thereby allow the cells to secrete extracellular matrix and to enable, in the long term, a complete and natural tissue replacement. The macromolecular structure of these biomaterials is selected so that they are completely degradable and are eliminated, once they have achieved their function of providing the initial artificial support for the remaining organ tissue cells. For these biomaterials to be useful in cell transplantations, they must be highly porous with large surface/volume ratios to accommodate a large number of cells, they must be biocompatible, i.e., non-toxic to the host tissue into which they are transplanted, they must be capable of promoting cell adhesion and allowing the retention of the differentiated function of attached cells.
Alginate is an anionic polysaccharide derived from brown algae. It is a block co-polymer of mannuronic acid (M) and guluronic acid (G). The polymer is widely used in the pharmaceutical, food and medical industries. The sodium salt of alginate is soluble in water and in the presence of divalent cations, such as calcium ions, it forms hydrogel at room temperature.
More specifically, alginates have been used previously for the purpose of cell transplantation. Alginates are natural polysaccharide polymers, the word “alginate” actually referring to a family of polyanionic polysaccharide copolymers derived from brown sea algae and comprising 1,4-linked P-D-mannuronic (M) and α-L-guluronic acid (G) residues in varying proportions. Alginates are soluble in aqueous solutions, at room temperature, and are capable of forming stable gels, particularly in the presence of certain divalent cations such as calcium, barium, and strontium. The unique properties of alginates, together with their biocompatibility [see Sennerby, L. et al. Biomaterials 8:49-52 (1987) and Cohen, S. et al. Proc. Natl. Acad. Sci. USA (In Press) 88(23):10440-10444 (1991)], relatively low cost and wide availability have made them important polymers in medicinal and pharmaceutical applications.
WO97/44070 by some of the present inventors, describes implantable polysaccharide, e.g. alginate sponges for use as a matrix, substrate or scaffold for the cultivation of mammalian cells in vitro prior to their implantation to replace damaged or removed tissue. WO2004/098669 also by part of the present inventors describes injectable cross-linked alginate, which forms a hydrogel in vivo. This cross-linked alginate solution was shown as effective in repair of cardiac tissue damage and ablation of cardiac arrhythmias, when locally applied onto the cardiac tissue.
Surprisingly, and in contrast, the present invention now shows that the alginate biomaterial as a solid, hydrogel, liquid (cross-linked as well as non-cross-linked) is by itself sufficient for the in vivo promotion of repair and regeneration of damaged tissue, decreasing the cellular damage and restoring organ synthetic functions to near-normal levels. Moreover, using non-cross-linked alginate solution, the invention demonstrates for the first time that systemic application, by an i.p. injection, leads to recovery of liver functions, and therefore is feasible for treating liver associated disorders.
The ability of the liver to regenerate itself enables it to overcome various forms of injuries [see Fausto, N. et al. Hepatology 43 (2Suppl 1):S45-53 (2006)]. Partial hepatectomy in humans is often needed and well tolerated in the setting of primary of secondary liver tumors [see Geller, D. A. et al. J. Gastrointest Surg. 10(1):63-8 (2006)]. Nevertheless, there are cases in which extended partial hepatectomy is warranted due to large hepatic mass and pose a high risk for fulminant hepatic failure [see Kubota, K. et al. Hepatology 26(5):1176-81 (1997)]. In these cases liver transplantation is the only option for treatment. To avoid this risk, innovative therapies such as portal vein embolization and staged liver resections have been both used, and were shown to be associated with considerable morbidity and mortality [see Madoff, D. C. et al. J. Vasc. Interv. Radiol. 16(6): 779-90 (2005); and Earle, S. A. et al. J. Am. Coll. Surg. 203(4):436-46 (2006)].
The alginate biomaterial solutions are ideal candidates for use as implants to facilitate restoration and regeneration of defective or missing tissues since they can be injected as low viscosity solution and can solidify on site [Landa, N. et al., Circulation 117:1388-1396 (2008)]. There is no need for additional curing methods, such as thermo-sensitive cross-linking or pH-sensitive cross-linking, photopolymerization, or addition of solidifying agents. It should be appreciated that most of these curing methods have drawbacks, for example, polymers that cure through a photopolymerization could pose a problem due to a limited ability to access the small cavities with light needed to initiate cross-linking.
Alginate biomaterials can further be manipulated by modification with adhesion peptides, such as RGD, to make them more adhesive, thus enhancing their interactions and integration with the host. For example, alginate modification with RGD reversed this polysaccharide from being cell-inert to a polysaccharide having cell adhesion promoting properties. Such modifications may be better interdigitate with the host [Tsur-Gang, O. et al., Biomaterials 30(2):189-195 (2009)]. In addition, biomaterials can be designed to enable the controlled delivery of therapeutic agents, such as growth factors and cytokines.
In search for efficient agents for in vivo, physical and functional repair of the damaged tissue, preferably an agent used systemically, the present inventors unexpectedly found that aqueous solutions of uncross-linked or calcium cross-linked alginate as well as solid forms of calcium-alginate hydrogels or sponges are able to facilitate functional restoration and regeneration of damaged liver following an extensive (90%) partial hepatectomy (PH) or following an immune mediated hepatitis.
It is thus an object of the present invention to provide a method for the treatment of liver damage and failure following extensive injury that employs a systemically injectable or in situ locally implantable biocompatible alginate biomaterial to support and regenerate the failing liver.
Another object of the invention to provide methods using alginate biomaterial in the treatment of subjects suffering from severe impairment of hepatic functions due to compulsory extensive partial hepatectomy or other acute or chronic liver disease.
These and other objects of the invention will become apparent as the description proceeds